Glucocorticoid-induced G1 arrest and the release effect of epidermal growth factor on the human salivary gland adenocarcinoma cell

Dexamethasone (1 microM) decreased the distribution of cells in S phase (about 75%) and increased that of G1 cells (1.1-fold) in the DNA histogram of human submandibular salivary gland adenocarcinoma cells (HSG) reversibly. In synchronized cells at G1 phase, glucocorticoid delayed the initiation of DNA synthesis by about 3-4 h. The conditioned medium (50%) or exogenous human epidermal growth factor (EGF, 10 ng/ml) significantly nullified these effects by glucocorticoids. These results suggested that glucocorticoids arrested the cells at G1 phase, which implied the inhibition of production of some progressive factor, probably EGF, in the cell cycle of HSG.     

The ERK-RSK1 activation by growth factors at G2 phase delays cell cycle progression and reduces mitotic aberrations

Growth factors accelerate G0 to S progression in the cell cycle, however, the roles of growth factors in other cell cycle phases are largely unknown. Here, we show that treatment of HeLa cells with hepatocyte growth factor (HGF) at G2 phase induced the G2/M transition delay as evidenced by FACS analysis as well as by mitotic index and time-lapse analyses. Growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) also induced G2/M transition delay like HGF. HGF treatment at G2 phase causes a delayed activation of cyclin B1-associated kinase and a diminished nuclear translocation of cyclin B1. Either U0126, a MAPK kinase (MEK) inhibitor, or kinase-dead mutant of ribosomal S6 kinase (RSK) abolished the delay. Additionally, knockdown of RSK1, but not RSK2, with siRNA abrogated the delay, indicating that the extracellular-regulated protein kinase (ERK)-RSK1 mediates the HGF-induced delay. We further found that the delay in G2/M transition of cells expressing oncogenic HGF receptor, M1268T, was abolished by RSK1 knockdown. Intriguingly, we observed that HGF induced chromosomal segregation defects, and depletion of RSK1, but not RSK2, aggravated these chromosomal aberrations. Taken together, the ERK-RSK1 activation by growth factors delays G2/M transition and this might be required to maintain genomic integrity during growth factor stimulation.     

Cross-linking of epidermal growth factor receptors in intact cells: detection of initial stages of receptor clustering and determination of molecular weight of high-affinity receptors

A method was developed to label epidermal growth factor (EGF) receptors with 125I-EGF in whole cells using chemical cross-linking reagents. Polyacrylamide gel electrophoresis resolved an Mr approximately 180,000 EGF-receptor complex and larger Mr greater than or equal to 360,000 aggregates. The formation of the larger complexes was time and temperature dependent and appeared to represent the initial events of EGF receptor clustering. Alteration of the ratio of 125I-EGF-labeled high- (Kd approximately 0.16 nM) and low- (Kd approximately 1.5 nM) affinity complexes by competition with unlabeled EGF or by induction of additional high-affinity sites with dexamethasone suggested that both sites were represented by the Mr approximately 180,000 125I-EGF-receptor complexes. Digestion of cells before cross-linking detected a small population of trypsin-resistant Mr approximately 180,000 receptors, which could represent previously described cryptic and/or high-affinity receptors. Few of the Mr approximately 360,000 receptors were trypsin resistant. Glucocorticoid induction of high-affinity EGF receptors failed to induce detectable changes in the microclustering of EGF receptors but did result in a 50% increase in EGF-induced receptor phosphorylation in HeLa S3 cell membranes at 4 degrees C. Thus, glucocorticoids increase high-affinity EGF binding sites, EGF-induced receptor phosphorylation, and cell growth.     

Cell cycle regulation of glucocorticoid receptor function.

Glucocorticoid receptor (GR) nuclear translocation, transactivation and phosphorylation were examined during the cell cycle in mouse L cell fibroblasts. Glucocorticoid-dependent transactivation of the mouse mammary tumor virus promoter was observed in G0 and S phase synchronized L cells, but not in G2 synchronized cells. G2 effects were selective on the glucocorticoid hormone signal transduction pathway, since glucocorticoid but not heavy metal induction of the endogenous Metallothionein-1 gene was also impaired in G2 synchronized cells. GRs that translocate to the nucleus of G2 synchronized cells in response to dexamethasone treatment were not efficiently retained there and redistributed to the cytoplasmic compartment. In contrast, GRs bound by the glucocorticoid antagonist RU486 were efficiently retained within nuclei of G2 synchronized cells. Inefficient nuclear retention was observed for both dexamethasone- and RU486-bound GRs in L cells that actively progress through G2 following release from an S phase arrest. Finally, site-specific alterations in GR phosphorylation were observed in G2 synchronized cells suggesting that cell cycle regulation of specific protein kinases and phosphatases could influence nuclear retention, recycling and transactivation activity of the GR.     

Nuclear antigens in the HeLa cell cycle

Summary Antisera to 0.35 M NaCl extracts and residues of S phase HeLa nuclei were reacted with electrophoretically separated proteins from the nuclei or nuclear material of HeLa cells synchronized in G₁, S, G₂ or M phases of the cell cycle. Quantitative evaluation of the peroxidase-antiperoxidase stained nitrocellulose transfers (Western blots) revealed significant changes in the quantities of nuclear non-histone proteins during the cell cycle. Immunochemical staining of electrophoretically separated nuclear antigens permits their selective detection in minute quantities and in the presence of many additional proteins.     

Mechanism of the glucocorticoid regulation of growth of the androgen-sensitive prostate-derived R3327H-G8-A1 tumor cell line

The R3327H-G8-A1 cell line derived from the Dunning rat prostate adenocarcinoma contains both androgen and glucocorticoid receptors. Following steroid deprivation, androgens specifically increase the concentration of their receptors in these cells by approximately 2-fold within 6 h and 3-4-fold in 24 h. In the presence of potent glucocorticoids, androgen receptor augmentation is reduced by 40-50% in the first 6 h and completely inhibited during the subsequent 24 h. This event, which is specific for glucocorticoids, appears to be due to an inhibition of androgen receptor synthesis. Furthermore, glucocorticoids inhibit proliferation of these cells by inhibiting the release of growth factors and arresting them in the G0 or A state of the cell cycle. This inhibition can be overcome by addition of low concentrations of either epidermal growth factor or platelet-derived growth factor; however, the inhibitory effect of the glucocorticoid on androgen receptor augmentation is not released. These results suggest that glucocorticoids arrest cellular proliferation by altering the autoregulation of growth and that this event is not dependent upon inhibition of androgen receptor augmentation.     

Objective identification of cell cycle phases and subphases by automated image analysis.

Frequency distributions of integrated optical density, perimeter, projection, area, form factor, average optical density, and mean dispersion path of nuclear images of Feulgen-stained HeLa S3 cells were obtained by automated image analysis at the base threshold of 0.04 OD. The mean values and standard deviations of these geometric parameters were then computed versus increasing values of threshold (0.08--0.32 OD). There is clear evidence of differential chromatin dispersion and convolution during the cycle of synchronized HeLa S3 cells at different times after selective mitotic detachment. The combination of average OD, form factor, and mean dispersion path at base threshold with the threshold dependence of nuclear morphometric parameters permits objective identification of cell cycle phases and their subphases, by characterizing variations in chromatin geometry within and between phases, regardless of whether DNA content remains constant (early G1, middle G1, late G1), varies only slightly (late G1-early S or late S-G2 transitions), or varies significantly (early S-middle S).     

cis-Diamminedichloroplatinum-mediated crosslinking of nuclear proteins to DNA is cell cycle specific

Immunochemical analysis was employed to investigate the cell cycle-dependent protein-DNA crosslinking by cis-diamminedichloroplatinum II (cis-DDP), in HeLa-S3 cells. Cells synchronized by double thymidine block or hydroxyurea were released into S phase and incubated at 2-h intervals with cis-DDP as they progressed through S1, G2, M, and then into G1 and S phases of the subsequent cycle. Immunoblots of the DNA-crosslinked antigens reacted with antisera to 0.35 M NaCl extract or residue of HeLa S-phase nuclei revealed that several antigens changed their DNA-crosslinking pattern during the progression of HeLa cells through their reproductive cycle.     

雌激素对兔子宫内膜细胞EGF受体和细胞周期的调控

 由受体放射配基结合分析证明家兔子宫内膜细胞的EGF受体Kd值为0.53nmol/L,每个细胞的最大结合容量为1.11×10~4结合位点。10~(-10)mol/L雌二醇处理24h,细胞的最大结合容量增至2.75×10~4结合位点数/细胞,而Kd值无明显变化,可是,当10~(-5)mol/L雌二醇处理24h,细胞的EGF受体结合率,DNA合成速度率均下降。G_0/G_1期细胞比值明显下降,而G_2+M期和S期细胞明显上升。     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

Fluctuation of trypsin-like proteinase activity in the cell cycle of synchronized and growing HeLa cells, and the effect of GMCHA-OPhBut

HeLa cells synchronized by double-thymidine block were grown in Eagle's minimum essential medium supplemented with 10% calf serum, and the fluctuation of trypsin-like protease activity in the cell cycle was examined. Seven distinct activity peaks were observed in one cell cycle at a cell density of 2%: two peaks in S phase, one peak at the S/G2 boundary, one peak in early M phase and one at the M/G1 boundary, and two peaks in G1 phase. HeLa cells synchronized by a mitotic detachment technique also showed similar results at cell density of 4.8%. The appearance of trypsin-like proteinase activity in the cell cycle was markedly affected by cell density, and no definite peak was observed above 8%. trans-Guanidinomethylcyclohexanecarboxylic and 4-tert-butylphenyl ester (GMCHA-OPhBut), a specific inhibitor for trypsin and a strong inhibitor of HeLa cell growth, had no effect on the various events in the first S, G2 and M phases, such as the incorporation of [methyl-3H]thymidine into DNA, the increase in the cell concentration, and the appearance of trypsin-like proteinase activity, whereas it retarded the onset of the second S phase and the various events in the second S, G2 and M phases for 3 h. In particular, it induced the appearance of a new proteinase peak at the G1/S boundary.     

Receptors for B-melanocyte-stimulating hormone exhibit positive cooperativity in synchronized melanoma cells

Cloudman S91 mouse melanoma cells respond in culture to B-melanocyte-stimulating hormone (B-MSH) with changes in morphology, growth rates, and melanin production. The effects of MSH appear to be mediated through a stimulation of the cyclic AMP system. It was reported earlier that at least some of the responses to MSH (increased cyclic AMP production and tyrosinase activity) occur in the G2 phase of the cell cycle [Wong, G., Pawelek, J., Sansone, M., & Morowitz, J. (1974) Nature (London) 248, 351-354] and that the apparent reason for this cell cycle restriction is that receptors for MSH are most active in the G2 phase [Varga, J. M., DiPasquale, A., Pawelek, J., McGuire, J., & Lerner, A. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1590-1593]. In this report, we found that by two separate methods of obtaining populations of cells in the G2 phase of their cycle--centrifugal elutriation or synchronization with thymidine--we observed increased binding of MSH by cells in the G2 and possibly late S phases of their cycle. However, cultures of cells passing through their cycle in synchrony were quite different from nonsynchronized (random) cultures. Both synchronized and random cultures expressed receptors for MSH in the G2 and possibly late S phases of their cycle, but synchronized cultures bound severalfold more MSH per cell than random cultures. This increased binding of MSH by synchronized cells was accompanied by an increase in tyrosinase activity and pigment production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Schlafen-1 causes a cell cycle arrest by inhibiting induction of cyclin D1

Schlafen-1 (Slfn-1), the prototypic member of the Schlafen family of proteins, was described as an inducer of growth arrest in T-lymphocytes and causes a cell cycle arrest in NIH3T3 fibroblasts prior to the G1/S transition. How Slfn-1 exerts its effects on the cell cycle is not currently known. We report that synchronized murine fibroblasts expressing Slfn-1 do not exit G1 when stimulated with fetal calf serum, platelet-derived growth factor BB (PDGF-BB) or epidermal growth factor (EGF). The induction of cyclin D1 by these stimuli was blocked in the presence of Slfn-1 as were all downstream cell cycle processes. Overexpression of cyclin D1 in growth-arrested, Slfn-1-expressing cells induced an increase in cell growth consistent with this protein being the biological target of Slfn-1. Activation of the mitogen-activated protein kinase pathway by EGF or phorbol 12-myristate 13-acetate was unaffected by Slfn-1 expression. PDGF signaling was, however, almost completely blocked. This was due to a lack of PDGF receptor expression in Slfn-1-expressing cells consistent with Slfn-1 blocking the cell cycle in G1 where PDGF receptor expression is normally down-regulated. Finally, overexpression of Slfn-1 inhibited the activation of the cyclin D1 promoter. Slfn-1 therefore causes a cell cycle arrest during G1 by inhibiting induction of cyclin D1 by mitogens.     

CENP—B的基因表达与细胞周期关系的研究

本文以HeLa细胞为材料研究一种着丝粒蛋白CENP-B的基因表达与细胞周期及细胞核骨架的关系。将HeLa细胞同步在不同周期时相,以流式细胞光度术、同位素掺入和ACA着丝粒染色等方法检测细胞同步化效果。我们分别提取了各周期时相细胞的总RNA和Poly(A)~ RNA,用Dot blot和Northern blot杂交方法研究CENP-B在细胞周期中的表达。结果表明,CENP-B基因在细胞周期中的各个时相均有表达,但表达的强度差别很大:G2期表达最强,S期最弱,G1期中的表达介于二者之间;有意义的是CENP-B基因在M期仍然有较强的表达,表现出其在细胞周期中表达的持续性;这种表达的持续性反映了一种可能性:着丝粒、动粒蛋白不断合成,但直到S期后进入G2期时着丝粒、动粒蛋白到一定临界浓度时才开始组装新的动粒。另外,着丝粒、动粒蛋白的持续合成对着丝粒、动粒功能的发挥可能是必需的。用Bam H I限制性内切酶消化处于不同细胞周期时相的HeLa细胞核骨架,提取与核骨架紧密结合的DNA,用~(32)P标记的cDNA为探针研究CENP-B基因与细胞核骨架的结合与其表达的关系。结果证明,在G2期细胞中CENP-B基因表达最强,与细胞核骨架结合最为紧密,G1期细胞中次之,S期中CENP-B基因与核骨架结合最弱,说明CENP-B基因与细胞核骨架结合的紧密度影响其表达强度。     

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

The physicochemical nature of 37 degrees C cytoplasmic glucocorticoid receptors in HeLa S3 cells

The physicochemical properties of size, shape and surface charge have been determined for the soluble fraction of cytoplasmic glucocorticoid receptors which are located in the HeLa S3 cell cytoplasm after incubation of whole cells with glucocorticoid at 37 degrees C. Under hypotonic buffer conditions approximately 80% of the total recovered [3H]triamcinolone acetonide receptor complexes sedimented through a 5-20% density gradients to the tube bottom, and approximately 90% eluted from a Sephacryl S-300 gel exclusion column in the void volume. Increasing the [KCl] of the buffer in the sucrose density gradients, and gel exclusion columns to 0.15 M caused a reduction in the percentage of this large aggregate to approximately 64% and approximately 75%, respectively. Further increases in the [KCl] during analysis to 0.4 M reduced the percentage of rapidly sedimenting receptors to approximately 62%, and shifted the sedimentation coefficient of the slower sedimenting receptors from approximately 5.2 S to 3.9 S. These conditions also decreased the fraction of receptor in the void volume of gel exclusion columns to 67%. Ion exchange analysis of receptor binding to DEAE cellulose, hydroxylapatite, phosphocellulose, and DNA cellulose revealed heterogenous populations of receptor species; comprising both "unactivated" and "activated" receptor forms. The ratios of unactivated/activated receptors was highly dependent on the matrix employed and differed substantially among those evaluated. For example, by the criteria of DEAE cellulose and phosphocellulose chromatography approximately 60% of the total 37 degrees C cytoplasmic receptors were in the "activated" state. A large fraction of these receptors, however, failed to bind to DNA cellulose. These results demonstrate that the glucocorticoid receptors which remain in the HeLa S3 cytoplasm at 37 degrees C do not bind to ion exchange materials, which are used as indexes of receptor "activation," in a uniform manner. We hypothesize that the diminished DNA binding capability of these receptors accounts for their cellular localization in the HeLa S3 cell cytoplasm at 37 degrees C.     

Characterization of glucocorticoid receptor in HeLa-S3 cells

Glucocorticoid receptor of the human cell line HeLa-S3 has been characterized and has been compared to rat and to mouse glucocorticoid receptors. If HeLa cells were lysed in the absence of glucocorticoid, glucocorticoid receptor was isolated in a nonactivated form, which did not bind to DNA-cellulose. If HeLa cells were preincubated with glucocorticoid, glucocorticoid receptor was isolated in an activated, DNA-binding form. HeLa cell glucocorticoid receptor bound [3H]triamcinolone acetonide with a dissociation constant (KD = 1.3 nM at 0 degrees C) that was similar to those of mouse and rat glucocorticoid receptors. Similarly, the relative binding affinities for steroid hormones decreased in the order of triamcinolone acetonide greater than dexamethasone greater than promegestone greater than methyltrienolone greater than aldosterone greater than or equal to moxestrol. Nonactivated and activated receptors were characterized by high-resolution anion-exchange chromatography (FPLC), DNA-cellulose chromatography, and sucrose gradient centrifugation. Human, mouse, and rat nonactivated glucocorticoid receptors had very similar ionic and sedimentation properties. Activated glucocorticoid receptors were eluted at similar salt concentrations from DNA-cellulose columns but at different salt concentrations from the FPLC column. A monoclonal mouse anti-rat liver glucocorticoid receptor antibody [Westphal, H.M., Mugele, K., Beato, M., & Gehring, U. (1984) EMBO J. 3, 1493-1498] did not cross-react with HeLa cell glucocorticoid receptor. Glucocorticoid receptors of HeLa, HTC, and S49.1 cells were affinity labeled with [3H]dexamethasone and with [3H]dexamethasone 21-mesylate. The molecular weights of [3H]dexamethasone 21-mesylate labeled glucocorticoid receptors (MT 96 000 +/- 1000) were undistinguishable by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor induction of phenotype-dependent cell cycle arrest in vascular smooth muscle cells is through the mitogen-activated protein kinase pathway

The heterogeneity of vascular smooth muscle cells is well established in tissue culture, but their differential responses to growth factors are not completely defined. We wished to identify effects of epidermal growth factor (EGF) on vascular smooth muscle cells in distinct phenotypes, such as spindle and epithelioid. We found that the EGF receptors were abundant in epithelioid cells but not spindle cells. EGF treatment inhibited serum-independent DNA synthesis, which was absent in spindle cells, of epithelioid cells. Additionally, using a pulse-chase assay, we found that bromodeoxyuridine-labeled cells failed to re-enter the S phase in the presence of EGF. These EGF effects were abolished by either inhibiting the EGF receptor tyrosine kinase with AG1478 or inhibiting the mitogen-activated protein kinase pathway with PD98059. In response to treatment with EGF, the EGF receptor was phosphorylated, which was correlated with phosphorylation and activation of p42/44 mitogen-activated protein kinases. Inhibition of EGF receptor phosphorylation and mitogen-activated protein kinase activation resulted in a reversal of the EGF-induced inhibition of bromodeoxyuridine incorporation and cell cycle arrest. Subsequent studies revealed that the activation of the EGF receptor and the mitogen-activated protein kinase pathway in epithelioid cells induced expression of the cell cycle inhibitory protein p27Kip1 but not p21Cip1. Taken together, our data demonstrate that the EGF receptor is abundantly expressed in epithelioid vascular smooth muscle cells and that the activation of this receptor results in cell cycle arrest through activation of the mitogen-activated protein kinase pathway.